Li Yu-Fen, Hsiao Yi-Hsiu, Lai Yi-Hui, Chen Yi-Chen, Chen Ying-Ju, Chou Jian-Liang, Chan Michael W Y, Lin Yu-Hsing, Tsou Yung-An, Tsai Ming-Hsui, Tai Chien-Kuo
a Institute of Biostatistics; China Medical University ; Taichung , Taiwan.
Epigenetics. 2015;10(3):229-36. doi: 10.1080/15592294.2015.1006506.
Oral squamous cell carcinoma (OSCC) constitutes >90% of oral cancers and is the sixth most common malignancy among males worldwide and the fourth leading cause of death due to cancer among males in Taiwan. However, most patients do not receive a diagnosis of OSCC until the late stages, which have a lower survival rate. The use of molecular marker analysis to identify early-stage OSCC would permit optimal timing for treatments and consequently prolong survival. The aim of this study was to identify biomarkers of OSCC using the Illumina GoldenGate Methylation Cancer Panel, which comprised a total of 1,505 CpG sites covering 807 genes. Samples of buccal mucosa resected from 40 OSCC patients and normal tissue samples obtained from 15 patients (normal mucosa from OSCC patients or from patients undergoing surgery unrelated to OSCC) were analyzed. Fms-related tyrosine kinase 4 (FLT4) methylation exhibited a perfect specificity for detecting OSCC, with an area under the receiver operating characteristic curve of 0.91 for both all-stage and early-stage OSCC. Methylation of 7 genes (ASCL1, FGF3, FLT4, GAS7, KDR, TERT, and TFPI2) constitutes the top-20 panels for detecting OSCC. The top-20 panels for detecting early-stage OSCC contain 8 genes: ADCYAP1, EPHA7, FLT4, GSTM2, KDR, MT1A, NPY, and TFPI2. FLT4 RNA expression and methylation level were validated using RT-PCR and a pyrosequencing methylation assay. The median level of FLT4 expression was 2.14-fold for normal relative to OSCC tissue samples (P < 0.0001). Among the 8 pyrosequenced FLT4 CpG sites, methylation level was much higher in the OSCC samples. In conclusion, methylation statuses of selected genes, and especially FLT4, KDR, and TFPI2, might be of great potential as biomarkers for early detection of buccal OSCC.
口腔鳞状细胞癌（OSCC）占口腔癌的90%以上，是全球男性中第六大常见恶性肿瘤，也是台湾男性癌症死亡的第四大主要原因。然而，大多数患者直到晚期才被诊断为OSCC，而晚期患者的生存率较低。使用分子标记分析来识别早期OSCC将有助于确定最佳治疗时机，从而延长生存期。本研究的目的是使用Illumina GoldenGate甲基化癌症检测板来识别OSCC的生物标志物，该检测板共包含1505个覆盖807个基因的CpG位点。对40例OSCC患者切除的颊黏膜样本和15例患者（OSCC患者的正常黏膜或接受与OSCC无关手术的患者的正常黏膜）获得的正常组织样本进行了分析。Fms相关酪氨酸激酶4（FLT4）甲基化对检测OSCC具有完美的特异性，在所有阶段和早期OSCC中，受试者操作特征曲线下面积均为0.91。七个基因（ASCL1、FGF3、FLT4、GAS7、KDR、TERT和TFPI2）的甲基化构成了检测OSCC的前20个检测板。检测早期OSCC的前20个检测板包含8个基因：ADCYAP1、EPHA7、FLT4、GSTM2、KDR、MT1A、NPY和TFPI2。使用RT-PCR和焦磷酸测序甲基化分析对FLT4 RNA表达和甲基化水平进行了验证。正常组织样本中FLT4表达的中位数水平相对于OSCC组织样本为2.14倍（P < 0.0001）。在8个经焦磷酸测序的FLT4 CpG位点中，OSCC样本中的甲基化水平要高得多。总之，所选基因的甲基化状态，尤其是FLT4、KDR和TFPI2，作为颊部OSCC早期检测的生物标志物可能具有巨大潜力。
